The paraventricular and posterior recess organs of elasmobranchs: A system of cerebrospinal fluid-contacting neurons containing immunoreactive serotonin and somatostatin

Summary The paraventricular organ (PVO) and the posterior recess organ (PRO) of two elasmobranch species, the spiny dogfish,Squalus acanthias, and the skate,Raja radiata, were investigated by use of scanning and transmission electron microscopy and immunocytochemistry employing a series of primary antisera. The PVO and PRO contained four types of cerebrospinal fluid (CSF)-contacting neurons. One type was free of secretory granules and projected a dendrite-like process into the ventricle. The other three types were distinguished according to the size of their secretory granules. The ventricular extensions of these cells were filled with secretory granules. By means of immunocytochemistry three types of CSF-contacting neurons were observed in the PVO and PRO. Type I contained only serotonin; type 2 displayed only somatostatin; type 3 was endowed with both serotonin and somatostatin. Type I dominated in the PRO, whereas type 3 was the most frequent in the PVO. The latter cells appear to be the site of origin of a loose tract formed by serotonin- and somatostatinimmunoreactive fibers projecting from the PVO into the neuropil of the PRO. Compact bundles formed exclusively by serotonin fibers were also shown to extend between the PVO and PRO. The basal processes of the CSF-contacting neurons of the PRO penetrated into the underlying neuropil. This neuropil is rich in synapses and can be regarded as an integrative area to which the basal processes of the local CSF-contacting neurons, serotonin and somatostatin fibers from the PVO, and fibers containing immunoreactive thyrotropin-releasing hormone of unknown origin, support a conspicuous input. The present findings indicate that the PVO and PRO of elasmobranchs are functionally integrated structures.Dedicated to Professor Erik Dahl on the occasion of his 75th birthday.     

Influence of thyroid hormone on the development of preoptic-hypothalamic monoaminergic neurons in tadpoles of Bufo bufo japonicus

The effect of thyroid hormone on the development of diencephalic monoaminergic neurons was studied in tadpoles of Bufo bufo japonicus. Monoamine-containing neurons in the preoptic recess organ (PRO) appeared later than those in the paraventricular organ (PVO) and nucleus infundibularis dorsalis (NID). After deprivation of thyroidal primordium no fluorescent neurons developed in the PRO. Development of monoaminergic neurons in the PVO and NID was not affected by thyroidectomy. Thyroxine treatment brought about the fluorescent neurons in the PRO of the thyroidectomized tadpoles. Fluorescent terminals in the median eminence became conspicuous around the capillaries which penetrated to the median emience, when the tadpoles reached late prometamorphic stage. In the median eminence of thyroidectomized tadpoles, the monoaminergic axon terminals did not develop. Thyroxine induced both the fluorescent terminals and the capillary penetration in the median eminence of the thyroidectomized tadpoles. In the tadpoles hypophysectomized at tail-bud stage, thyroxine induced neither the fluorescent terminals nor the capillaries in the median eminence.     

Photoperiodic response of serotonin- and galanin-immunoreactive neurons of the paraventricular organ and infundibular nucleus in Japanese quail, Coturnix coturnix japonica

We investigated the photoperiodic response of serotonin- and galanin (GA)- immunoreactive (ir) cells in the paraventricular organ (PVO) and infundibular nucleus (IF) of the Japanese quail and the interaction of these cells with gonadotropin-releasing hormone (GnRH)-ir neurons in the hypothalamus. Serotonin-ir cells were located in series from the PVO to the IF, and were connected with each other. The number of serotonin-ir cells differed significantly between light and dark phases on the short days (SD), but did not differ between light and dark phases on long days (LD). GA-ir cells were also found in the PVO and IF. The number of GA-ir cells under SD conditions was significantly greater than under LD conditions but did not change diurnally. Both serotonin-ir and GA-ir fibers ran along the GnRH-ir cells in the nucleus commissurae pallii. Serotonin-ir and GA-ir fibers were connected with the GnRH-ir fibers in the external layer of the median eminence (ME). We confirmed that GA-ir fibers were closely associated with serotonin-ir neurons in the PVO and IF. GA-ir neurons have at least 2 routes of regulating GnRH neurons directly, and indirectly via the serotonin-ir cells in the PVO and IF.     

Immunocytochemistry of luteinizing hormone-releasing hormone in brains of bullfrogs (Rana catesbeiana) during spontaneous metamorphosis

The distribution of immunoreactive luteinizing hormone-releasing hormone (ir-LH-RH) in brains of bullfrogs (Rana catesbeiana) during spontaneous metamorphosis has been studied by combination of an unlabeled antibody enzyme immunocytochemical technique and an adjacent serial section approach. In prometamorphic tadpoles, immunocytochemical staining for ir-LH-RH was absent from the brain, including a structurally simple median eminence (ME) and perikarya in the anterior preoptic area (aPOA). In metamorphic tadpoles, speckled patches of immunostaining occurred over the outer layer of a modestly developed ME; coincident faint staining of a small number of medial, unpaired cell bodies was localized in the aPOA. In newly metamorphosed juvenile frogs, more diffuse and intense staining of the outer layer of the ME accompanied increased morphological differentiation of this neurohemal area; immunoreactive perikarya again were found in the aPOA, but an increased number of neurons exhibited comparatively greater (moderate) immunostaining. Changes in the quality of immunostaining and in the numbers of cells stained, therefore, were coincident with metamorphic development. Concomitant alterations of ir-LH-RH immunostaining and progressive structural development of the ME suggest a coordinated differentiation of brain neuroendocrine systems during metamorphosis of the bullfrog tadpole.     

Distribution of catecholaminergic and serotoninergic systems in forebrain and midbrain of the newt,Triturus alpestris (Urodela)

Summary Mapping of monoaminergic systems in the brain of the newt Triturus alpestris was achieved with antisera against (1) thyrosine hydroxylase (TH), (2) formaldehyde-conjugated dopamine (DA), and (3) formaldehyde-conjugated serotonin (5-HT). In the telencephalon, the striatum was densely innervated by a large number of 5-HT-, DA-and TH-immunoreactive (IR) fibers; IR fibers were more scattered in the amygdala, the medial and lateral forebrain bundles, and the anterior commissure. In the anterior and medial diencephalon, TH-IR perikarya contacting the cerebrospinal fluid (CSF-C perikarya) were located in the preoptic recess organ (PRO), the organum vasculosum laminae terminalis and the suprachiasmatic nucleus. Numerous TH-IR perikarya, not contacting the CSF, were present in the posterior preoptic nucleus and the ventral thalamus. At this level, DA-IR CSF-C neurons were only located in the PRO. In the posterior diencephalon, large populations of 5-HT-IR and DA-IR CSF-C perikarya were found in the paraventricular organ (PVO) and the nucleus infundibularis dorsalis (NID); the dorsal part of the NID additionally presented TH-IR CSF-C perikarya. Most regions of the diencephalon showed an intense monoaminergic innervation. In addition, numerous TH-IR, DA-IR and 5-HT-IR fibers, orginating from the anterior and posterior hypothalamic nuclei, extended ventrally and reached the median eminence and the pars intermedia of the pituitary gland. In the midbrain, TH-IR perikarya were located dorsally in the pretectal area. Ventrally, a large group of TH-IR cell bodies and some weakly stained DA-IR and 5-HT-IR neurons were observed in the posterior tuberculum. No dopaminergic system equivalent to the substantia nigra was revealed. The possible significance of the differences in the distribution of TH-IR and DA-IR neurons is discussed, with special reference to the CSF-C neurons.Abbreviations AM amygdala - CAnt commissura anterior - CH commissura hippocampi - CP commissura posterior - Ctm commissura tecti mesencephali - DH dorsal hypothalamus - DTh dorsal thalamus - FLM fasciculus longitudinalis medialis - Fsol fasciculus solitarius - H habenula - LFB lateral forebrain bundle - ME median eminence - MFB medial forebrain bundle - NID nucleus infundibularis dorsalis - nIP neuropil of nucleus interpeduncularis - NPOP nucleus preopticus posterior - NS nucleus septi - OVLT organum vasculosum laminae terminalis - PD pars distalis - Pdo dorsal pallium - PHi primordium hippocampi - PI pars intermedia - Pl lateral pallium - PN pars nervosa - PRO preoptic recess organ - Ptec pretectal area - PVO paraventricular organ - Ra nucleus raphe - Rm nucleus reticularis medius - SCO subcommisural organ - ST striatum; strm stria medullaris thalami - strt stria terminalis thalami - TM tegmentum mesencephali - TO tectum opticum - TP tuberculum posterius - trch tractus cortico-habenularis - trmp tractus mamillopeduncularis - VH ventral hypothalamus - Vm nucleus motorius nervi trigemini - VTh ventral thalamus - II optic nerve     

Distribution of lamprey gonadotropin-releasing hormone-III (GnRH-III) in brains of larval lampreys (Petromyzon marinus)

Two immunoreactive forms of gonadotropinreleasing hormone (GnRH), lamprey GnRH-I and lamprey GnRH-III, were found in neurons in larval sea lampreys (Petromyzon marinus). Using antisera preferentially directed against either lamprey GnRH-I or-III, dense reaction product was seen in cell bodies in the rostral hypothalamus and preoptic area. Reaction product was also dense in fibers to and within the neurohypophysis, in addition to numerous fibers which projected caudally, beyond the neurohypophysis through the mesencephalon. The majority of immunoreactive GnRH was lamprey GnRH-III, and when lamprey GnRH-I was seen, it was in cells that appeared to contain both forms of GnRH. A small number of cells found in the caudal hypothalamus contained only immunoreactive lamprey GnRH-III, and these may constitute a functional subgroup within the population of GnRH neurons. In animals undergoing metamorphosis there was a large increase in reaction product in all GnRH-containing cells and fibers. A striking change within the distribution of GnRH cells was localized to a distinct group of GnRH-immunoreactive cells (GnRH-I and-III) in the ventral anterior hypothalamic area. These cells were minimally detectable in larvae, but during metamorphosis became densely filled with immunoreactive product in perikarya and distal processes. The results are consistent with the hypothesis that lamprey GnRH-III is an important form of GnRH during the maturation of GnRH cells and fibers, and further indicates that these cells have attained their normal positions in the preoptic area and hypothalamus before metamorphosis.     

Distribution of LHRH in the rat and mouse brain with special reference to the tanycytes

Summary The distribution of luteinizing hormone-releasing hormone (LHRH) was studied in the rat and mouse brain by means of light and electron microscopic immunohistochemistry using the peroxidase-antiperoxidase method. An immunoreactive product to LHRH antiserum was found near the blood vessels of the vascular organ of the lamina terminalis. In the arcuate nucleus-median eminence region, an immunoreactive material occurred bilaterally in the hypothalamic tissue around the tuberoinfundibular sulci. Electron microscopy revealed that immunoreactive fibers observed light microscopically contain numerous granules 100–130 nm in diameter. No immunoreactive product was located in the tanycytes of the median eminence, the perikarya of hypothalamic neurons, and the parenchyma of several circumventricular organs (subfornical organ, subcommissural organ, pineal organ, area postrema).Supported by grants from the Ministry of Education of Japan and the Ford Foundation     

Neural interaction of gonadotropin-regulating hormone immunoreactive neurons and the suprachiasmatic nucleus with the paraventricular organ in the Japanese grass lizard (Takydromus tachydromoides)

Our previous study demonstrated that the paraventricular organ (PVO) in the hypothalamus of the Japanese grass lizard (Takydromus tachydromoides) showed immunoreactivity against the light signal-transducing G-protein, transducin. This finding suggested that the PVO was a candidate for the deep-brain photoreceptor in this species. To understand functions of the PVO, we investigated distributions of transducin, serotonin, gonadotropin-releasing hormone (GnRH), and gonadotropin-inhibitory hormone (GnIH) in the lizard's brain. We immunohistochemically confirmed co-localization of transducin and serotonin in PVO neurons that showed structural characteristics of cerebrospinal fluid (CSF)-contacting neurons. GnRH-immunoreactive (ir) cells were localized in the posterior commissure and lateral hypothalamic area. Some of the serotonin-ir fibers extending from the PVO to the lateral hypothalamic area contacted the GnRH-ir cell bodies. GnIH-ir cells were localized in the nucleus accumbens, paraventricular nucleus, and upper medulla, and GnIH-ir fibers from the paraventricular nucleus contacted the lateral processes of serotonin-ir neurons in the PVO. In addition, we found that serotonin-ir fibers from the PVO extended to the suprachiasmatic nucleus (SCN), and the retrograde transport method confirmed the PVO projections to the SCN. These findings suggest that the PVO, by means of innervation mediated by serotonin, plays an important role in the regulation of pituitary function and the biological clock in the Japanese grass lizard.     

Nigrostriatal Modifications After Vanadium Inhalation: An Immunocytochemical and Cytological Approach

Vanadium (V) has increased in the air as a component of suspended particles originated from fuel combustion. In this report, a model of inhaled V in mice was implemented to identify the effect that V has in the corpus striatum and substantia nigra, structures with high concentrations of dopamine and scarce antioxidants burden. Mice inhaled 0.02 M V2O5 1 h twice a week and were sacrificed at points from 1 to 8 weeks after inhalation, perfused, and processed for Golgi method and for tyroxine hidroxylase (TH) inmunocytochemistry. Cytological analysis consisted in counting the number of dendritic spines in 20 medium-size spiny neurons and the number of TH immunoreactive neurons in the substatia nigra pars compacta. Dendritic spine density decreased drastically after V exposure; the same was observed with the TH-positive neurons, which decreased in a time-dependent mode. No previous morphological studies about V and nervous system have been reported. The decrease in spine density and in TH-positive neurons might have functional repercussions that should be studied because the trend of this element in the atmosphere is to increase.     

Possible involvement of brain-derived neurotrophic factor (BDNF) in the innervation of dopaminergic neurons from the rat periventricular nucleus to the pars intermedia

Developing neurons are guided to their appropriate targets by specific guidance substances that have neurotrophic actions. The aim of the present study was to elucidate the mechanism by which hypothalamic neurons reach the pars intermedia (PI) by correlating the development of dopaminergic (DA) neurons arising in the periventricular nucleus (PeV) of fetal rats with the expression of brain-derived neurotrophic factor (BDNF) in the rat pituitary. The differentiation of DA neurons was observed by immunohistochemistry using an antibody against tyrosine hydroxylase (TH), whereas the ontogenesis of BDNF mRNA in the PI was examined by in situ hybridization and RT-PCR. Immunoreactive TH-neurons were first observed in the PeV at embryonic day (E) 16.5, following which time their axons elongated toward the pituitary. TH-positive reactions were observed in the connective tissue between the PI and the pars nervosa at E20.5. Innervation of the PI by TH-positive neurons was determined at postnatal day (P) 1.5; however, BDNF mRNA was first detected in the PI cells at E17.5, with an increase in its expression clearly visible at E21.5 and continuing high expression levels in the PI thereafter. These results suggest that BDNF is a specific guidance cue for DA neurons elongating from the PeV to the PI.     

Organization of tyrosine hydroxylase-immunoreactive neurons in the di-and mesencephalon of the American bullfrog (Rana catesbeiana) during metamorphosis

Summary We examined the immunocytochemical distribution of tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis, in the di-and mesencephalon of developing bullfrog tadpoles. Special attention was given to catecholaminergic innervation of the median eminence and pituitary. In premetamorphic tadpoles, tyrosine hydroxylase-immunoreactive neurons were visualized in the suprachiasmatic and infundibular hypothalamus, the ventral thalamus, and midbrain tegmentum by Taylor-Kollros stage V. The number of labeled neurons in all these areas increased as metamorphosis progressed. By mid-prometamorphosis, labeled neurons appeared in the preoptic recess organ as well as in the posterior thalamic nucleus. The majority of cells in the preoptic recess organ, as well as occasional neurons in the suprachiasmatic nucleus, exhibited labeled processes which projected through the ependymal lining of the preoptic recess to contact cerebrospinal fluid. The modified CSF-contacting neurons of the nucleus of the periventricular organ were devoid of specific staining. By late prometamorphosis, labeled fibers from the suprachiasmatic nucleus were observed projecting caudally to enter the hypothalamo-hypophysial-tract en route to innervating the median eminence and pituitary. Labeled fibers arising from the dorsal infundibular nucleus projected ventrolaterally to contribute to catecholaminergic innervation of the median eminence and pituitary. Immunoperoxidase staining of tyrosine hydroxylase-immunoreactive fibers and terminal arborizations in the median eminence were restricted to non-ependymal layers, while labeled fibers in the pituitary were observed in the pars intermedia and pars nervosa. Staining of tyrosine hydroxylase-immunoreactive fibers in the median eminence and pituitary was sparse or absent in premetamorphic tadpoles, but became increasingly more intense as metamorphosis progressed.     

Development of hypothalamic neurons in intraventricular grafts: expression of specific transmitter phenotypes

The anlages of the medial-basal hypothalamus (MBH), septopreoptic area (POA), Rathke's pouch, and the parietal cortex (CC) of rats (at 12.5, 14.5 and 16.5 days of gestation) were transplanted singly or in combination into the third ventricle of adult female rats, and the development of neurons in the grafts was investigated immunohistochemically with the use of antisera to tyrosine hydroxylase (TH), somatostatin (SRIH), ACTH, methionine enkephalin-Arg6-Gly7-Leu8 (Enk-8), rat corticotropin-releasing factor (rCRF), rat hypothalamic growth hormone-releasing factor (rhGRF), and luteinizing hormone-releasing hormone (LHRH). TH and all the peptides examined except LHRH were detected in distinct neurons in MBH grafts and in cografts of MBH plus Rathke's pouch from 12.5-day-old embryos. SRIH, rCRF, Enk-8, and TH were found in POA grafts from embryos of the same age. Although immunoreactive LHRH was first detected in neurons in POA grafts from 16.5-day-old embryos, it appeared in cografts of POA and MBH from 12.5-day-old embryos. The immunoreactive fibers developed in the grafts expressed the same characteristic behaviors as in intact brain; the fibers containing hormonal substances formed complexes with the vasculature like in the organum vasculosum laminae terminalis (OVLT) or in the median eminence, while the fibers containing neurotropic signals formed fiber networks surrounding other nerve cell bodies as if they synaptically associate. In CC grafts, the neurons contained TH, SRIH, rCRF, or Enk-8, and their axonal processes formed fiber networks. These findings suggest that all the hypothalamic neurons examined are committed by 12.5 days of gestation to develop maintaining transmitter phenotype and target recognition capacity.     

A histochemical study of the primary catecholamines in the hypothalamic neurons of the rat in relation to the ontogenetic and sexual differentiation

Summary The alterations in the content of the primary catecholamines in the hypothalamus have been studied with the histochemical technique of para-formaldehyde induced fluorescence.In the adult normal rats, independent of the sex, the fluorescence is located in the cell bodies of a few arcuate neurons, around the perikarya of the arcuate, para-ventricular and supra-optic neurons, and in the nerve endings of the arcuate neurons in the median eminence.The appearance of the primary catecholamines takes place at the 20th day of gestation in the para-ventricular and arcuate-peri-ventricular regions. In the supra-optic nucleus the fluorescent nerve terminals are not seen before birth. In the outer layer of the median eminence the fluorescence develops around the 5th post-natal day. No sexual differences were observed in the maturation of the primary catecholamines during the ontogenic development of the rat.More fluorescent cell bodies and nerve endings are seen in the arcuate neurons during the late diestrus than during estrus. The number and intensity of the catecholamine fluorescent neurons in the arcuate nucleus increases during the pregnancy. Castration increases slightly the number and intensity of the fluorescent cell bodies in the arcuate nucleus, but it diminishes the fluorescence in the median eminence. The changes were compensated by a treatment with testosterone propionate. Hypophysectomy alone has no effect on the fluorescence of the hypothalamic neurons.Supported by a grant from The Finnish Medical Society Duodecim.     

Three Types of Tyrosine Hydroxylase-Positive CNS Neurons Distinguished by Dopa Decarboxylase and VMAT2 Co-Expression

Sumary 1. We investigate here for the first time in primate brain the combinatorial expression of the three major functionally relevant proteins for catecholaminergic neurotransmission tyrosine hydroxylase (TH), aromatic acid acid decarboxylase (AADC), and the brain-specific isoform of the vesicular monoamine transporter, VMAT2, using highly specific antibodies and immunofluorescence with confocal microscopy to visualize combinatorial expression of these proteins.2. In addition to classical TH, AADC, and VMAT2-copositive catecholaminergic neurons, two unique kinds of TH-positive neurons were identified based on co-expression of AADC and VMAT2.3. TH and AADC co-positive, but VMAT2-negative neurons, are termed “nonexocytotic catecholaminergic TH neurons.” These were found in striatum, olfactory bulb, cerebral cortex, area postrema, nucleus tractus solitarius, and in the dorsal motor nucleus of the vagus.4. TH-positive neurons expressing neither AADC nor VMAT2 are termed “dopaergic TH neurons.” We identified these neurons in supraoptic, paraventricular and periventricular hypothalamic nuclei, thalamic paraventicular nucleus, habenula, parabrachial nucleus, cerebral cortex and spinal cord. We were unable to identify any dopaergic (TH-positive, AADC-negative) neurons that expressed VMAT2, suggesting that regulatory mechanisms exist for shutting off VMAT2 expression in neurons that fail to biosynthesize its substrates.5. In several cases, the corresponding TH phenotypes were identified in the adult rat, suggesting that this rodent is an appropriate experimental model for further investigation of these TH-positive neuronal cell groups in the adult central nervous system. Thus, no examples of TH and VMAT2 co-positive neurons lacking AADC expression were found in rodent adult nervous system.6. In conclusion, the adult mammalian nervous system contains in addition to classical catecholaminergic neurons, cells that can synthesize dopamine, but cannot transport and store it in synaptic vesicles, and neurons that can synthesize only L-dopa and lack VMAT2 expression. The presence of these additional populations of TH-positive neurons in the adult primate CNS has implications for functional catecholamine neurotransmission, its derangement in disease and drug abuse, and its rescue by gene therapeutic maneuvers in neurodegenerative diseases such as Parkinson's disease.     

Monoamine fluorescence in the median eminence of the Japanese quail,Coturnix coturnix japonica,following medial basal hypothalamic deafferentation

Summary Monoamine fluorescence was examined in the ventral hypothalamus of the Japanese quail, Coturnix coturnix japonica after medial basal hypothalamic deafferentation. In sham-operated control birds, numerous yellow-green fluorescent fibers were observed in the median eminence and the nucleus tuberis. In the area of the paraventricular organ, a number of fluorescent fibers and cell bodies were observed. In birds with deafferented hypothalami, fluorescence disappeared both in the median eminence and the nucleus tuberis. In the area of the paraventricular organ, which was within the area of deafferentation, fluorescence of neuronal perikarya did not change, but fluorescent fibers decreased markedly in number. Disappearance of monoamine fluorescence in the median eminence and the nucleus tuberis is discussed in relation to the tanycyte absorptive function and gonadal development.Supported by Grants from the Ministry of Education to Professors T. Bando and H. Kobayashi, and a Grant from the Ford Foundation to Prof. H. Kobayashi.     

Identification of neuron types in the submucosal ganglia of the mouse ileum

The continuing and even expanding use of genetically modified mice to investigate the normal physiology and development of the enteric nervous system and for the study of pathophysiology in mouse models emphasises the need to identify all the neuron types and their functional roles in mice. An investigation that chemically and morphologically defined all the major neuron types with cell bodies in myenteric ganglia of the mouse small intestine was recently completed. The present study was aimed at the submucosal ganglia, with the purpose of similarly identifying the major neuron types with cell bodies in these ganglia. We found that the submucosal neurons could be divided into three major groups: neurons with vasoactive intestinal peptide (VIP) immunoreactivity (51% of neurons), neurons with choline acetyltransferase (ChAT) immunoreactivity (41% of neurons) and neurons that expressed neither of these markers. Most VIP neurons contained neuropeptide Y (NPY) and about 40% were immunoreactive for tyrosine hydroxylase (TH); 22% of all submucosal neurons were TH/VIP. VIP-immunoreactive nerve terminals in the mucosa were weakly immunoreactive for TH but separate populations of TH- and VIP-immunoreactive axons innervated the arterioles in the submucosa. Of the ChAT neurons, about half were immunoreactive for both somatostatin and calcitonin gene-related peptide (CGRP). Calretinin immunoreactivity occurred in over 90% of neurons, including the VIP neurons. The submucosal ganglia and submucosal arterioles were innervated by sympathetic noradrenergic neurons that were immunoreactive for TH and NPY; no VIP and few calretinin fibres innervated submucosal neurons. We conclude that the submucosal ganglia contain cell bodies of VIP/NPY/TH/calretinin non-cholinergic secretomotor neurons, VIP/NPY/calretinin vasodilator neurons, ChAT/CGRP/somatostatin/calretinin cholinergic secretomotor neurons and small populations of cholinergic and non-cholinergic neurons whose targets have yet to be identified. No evidence for the presence of type-II putative intrinsic primary afferent neurons was found. This work was supported by a grant from the National Health and Medical Research Council of Australia (grant no. 400020) and an Australian Research Council international linkage grant (no. LZ0882269) for collaboration between the Melbourne and Bologna laboratories.     

Vasoactive intestinal polypeptide (VIP)-containing neurons: distribution throughout the brain of the chick (Gallus domesticus) with focus upon the lateral septal organ

The distribution of VIP-like perikarya and fibers was determined throughout the chick brain. The most rostral immunoreactive perikarya were found to be cerebrospinal fluid-contacting neurons in the pars medialis of the lateral septal organ. Additional data were presented supporting the idea that the lateral septal organ is another circumventricular organ within the brain of birds (Kuenzel and van Tienhoven 1982). A large group of immunoreactive perikarya was found in the lateral hypothalamic area and appeared continuous with immunoreactive neurons in the anterior medial and ventromedial hypothalamic nuclei (n). A few perikarya were located in the paraventricular hypothalamic n. A number of immunoreactive neurons were found within and about the infundibular and inferior hypothalamic n., none however was immunoreactive cerebrospinal fluid-contacting neurons. Immunoreactive perikarya were found predominantly in laminae 10–11 of the stratum griseum et fibrosum superficiale. A few scattered perikarya were found ventromedial to the n. tegmenti pedunculo-pontinus pars compacta and locus ceruleus. Some of the immunoreactivity was unusual, being very homogeneous within the cell body with little evidence of the material in the axon or dendrites. Perikarya were found in the central gray, n. intercollicularis, and area ventralis of Tsai. The most caudal structure showing immunoreactive neurons was the n. reticularis paragigantocellularis lateralis. Brain areas containing the most abundant immunoreactive fibers, listed from the rostral-most location, were found in the ventromedial region of the lobus parolfactorius and the lateral septal n. Continuing caudally, there were immunoreactive fibers within the periventricular hypothalamic n.; some of the fibers were found to travel for some distance parallel to the third ventricle. Dense immunoreactive fibers were found in the tractus cortico-habenularis et cortico-septalis, medial habenular n. and posterior and dorsal n. of the archistriatum. A number of areas had what appeared to be baskets of immunoreactive fibers (perhaps immunoreactive terminals) surrounding non-reactive perikarya. Brain areas containing terminals included the piriform cortex, area ventralis of Tsai, interpeduncular n., and specific regions of the stratum griseum et fibrosum superficiale. A very dense immunoreactivity occurred within the external zone of the median eminence, the dorsolateral parabrachial n., and n. tractus solitarii. Vasoactive intestinal polypeptide appears to be a useful peptide for defining the neuroanatomical constituents of the visceral forebrain in birds.     

An attempt to record neuronal activity in the paraventricular organ of Rana esculenta by means of a direct access to the infundibular recess

Summary In submammalian vertebrates, the paraventricular organ (PVO) of the third ventricle is a complex circumventricular structure composed of cerebrospinal fluid-contacting neurons and corresponding deeper formations of nerve cells. A new in-vivo technique enables us to approach the paraventricular organ of the frog, Rana esculenta, via the lobus infundibularis. In this preparation, blood flow in the capillary loops beneath the PVO and the flow of the cerebrospinal fluid in the infundibular recess can be directly observed. Electrical recordings of neural activity in and near the PVO show continuous and phasic, spontaneous activity. Light stimulation of the retina and direct illumination of the brain were not followed by alterations of nerve cell activity. A major problem in the electrophysiological investigation is the diminution in spontaneous activity of the recorded neurons after exchange of CSF.     

Dopamine D1 and D2 Receptor Immunoreactivities in the Arcuate-Median Eminence Complex and their Link to the Tubero-Infundibular Dopamine Neurons

Dopamine D1 and D2 receptor immunohistochemistry and Golgi techniques were used to study the structure of the adult rat arcuate-median eminence complex, and determine the distribution of the dopamine D1 and D2 receptor immunoreactivities therein, particularly in relation to the tubero-infundibular dopamine neurons. Punctate dopamine D1 and D2 receptor immunoreactivities, likely located on nerve terminals, were enriched in the lateral palisade zone built up of nerve terminals, while the densities were low to modest in the medial palisade zone. A codistribution of dopamine D1 receptor or dopamine D2 receptor immunoreactive puncta with tyrosine hydroxylase immunoreactive nerve terminals was demonstrated in the external layer. Dopamine D1 receptor but not dopamine D2 receptor immnunoreactivites nerve cell bodies were found in the ventromedial part of the arcuate nucleus and in the lateral part of the internal layer of the median eminence forming a continuous cell mass presumably representing neuropeptide Y immunoreactive nerve cell bodies. The major arcuate dopamine/ tyrosine hydroxylase nerve cell group was found in the dorsomedial part. A large number of tyrosine hydroxylase immunoreactive nerve cell bodies in this region demonstrated punctate dopamine D1 receptor immunoreactivity but only a few presented dopamine D2 receptor immunoreactivity which were mainly found in a substantial number of tyrosine hydroxylase cell bodies of the ventral periventricular hypothalamic nucleus, also belonging to the tuberoinfundibular dopamine neurons. Structural evidence for projections of the arcuate nerve cells into the median eminence was also obtained. Distal axons formed horizontal axons in the internal layer issuing a variable number of collaterals classified into single or multiple strands located in the external layer increasing our understanding of the dopamine nerve terminal networks in this region. Dopamine D1 and D2 receptors may therefore directly and differentially modulate the activity and/or Dopamine synthesis of substantial numbers of tubero-infundibular dopamine neurons at the somatic and terminal level. The immunohistochemical work also gives support to the view that dopamine D1 receptors and/or dopamine D2 receptors in the lateral palisade zone by mediating dopamine volume transmission may contribute to the inhibition of luteinizing hormone releasing hormone release from nerve terminals in this region.Key words: Dopamine D1 and D2 receptors, tubero-infundibular dopamine neurons, dopamine receptor colocalization, arcuate-median eminence complex, volume transmission, luteinizing hormone releasing hormone     

Immunocytochemical localization of neuropeptide Y-like immunoreactivity in adrenergic and non-adrenergic neurons of the rat gastrointestinal tract

The immunocytochemical location of neuropeptide Y (NPY)-like immunoreactivity (LI) within the neuronal structures of the rat gastrointestinal (GI) tract was investigated with the indirect immunofluorescence method. NPY immunoreactive neurons were found throughout all regions of the GI tract with the largest number in the duodenum. NPY immunoreactive perikarya were mainly located in the submucosal ganglia. NPY labeled processes were extensively seen in the submucosal and myenteric plexuses, smooth muscles, muscularis mucosa, mucosa and surrounding blood vessels. Following 6-hydroxydopamine (6-OHDA) treatment, NPY immunoreactive nerve fibers around blood vessels disappeared completely and the reactive fibers in other regions were reduced in number. NPY immunoreactive nerve cell bodies in the ganglionic plexuses, however, were not affected by 6-OHDA treatment. Serial sections of the coeliac ganglion showed that NPY-LI was present in cell bodies which also displayed tyrosine hydroxylase (TH) immunoreactivity. Our results suggest that NPY is abundantly contained in both adrenergic and non-adrenergic neurons of the gut and may play an important role in the regulation of the GI tract.